Self-assembling peptide and peptide gel with high strength

ABSTRACT

Provide are a peptide gel with practically sufficient mechanical strength and a self-assembling peptide capable of forming the peptide gel. The self-assembling peptide is formed of the following amino acid sequence: a 1 b 1 c 1 b 2 a 2 b 3  db 4 a 3 b 5 c 2 b 6 a 4  where: a 1  to a 4  each represent a basic amino acid residue; b 1  to b 6  each represent an uncharged polar amino acid residue and/or a hydrophobic amino acid residue, provided that at least five thereof each represent a hydrophobic amino acid residue; c 1  and c 2  each represent an acidic amino acid residue; and d represents a hydrophobic amino acid residue.

TECHNICAL FIELD

The present invention relates to a self-assembling peptide capable offorming a high-strength peptide gel, and a peptide gel, which is formedfrom the peptide.

BACKGROUND ART

A collagen gel is generally used as a scaffold (scaffold for cells) tobe used in research and actual therapy in the regenerative medicinefield. However, the collagen gel is derived from an animal and hence maycause an unknown infectious disease. As means for eliminating theconcern about the unknown infectious disease, there exists a scaffoldderived from a chemically synthesized material. Examples of suchmaterial include a self-assembling peptide disclosed in Patent Document1 or Patent Document 2. However, a scaffold (peptide gel) formed of theself-assembling peptide of Patent Document 1 or 2 is insufficient inmechanical strength and hence involves such a problem in handleabilitythat the scaffold may collapse when grasped with tweezers, for example.Further, the self-assembling peptide gel of Patent Document 1 involvessuch a problem that the gel is insufficient in transparency at neutralpH environment.

PRIOR ART DOCUMENTS Patent Documents

-   [Patent Document 1] U.S. Pat. No. 5,670,483 A-   [Patent Document 2] WO 2007/000979 A1

SUMMARY OF INVENTION Problems to be Solved by the Invention

The present invention has been made in order to solve theabove-mentioned problem. An object of the present invention is toprovide a peptide gel with practically sufficient mechanical strengthand a self-assembling peptide capable of forming the peptide gel.

Means for Solving the Problems

According to the present invention, a self-assembling peptide isprovided. The self-assembling peptide is formed of the following aminoacid sequence:

-   -   Amino acid sequence: a₁b₁c₁b₂a₂b₃ db₄a₃b₅c₂b₆a₄        where: a₁ to a₄ each represent a basic amino acid residue; b₁ to        b₆ each represent an uncharged polar amino acid residue and/or a        hydrophobic amino acid residue, provided that at least five        thereof each represent a hydrophobic amino acid residue; c₁ and        c₂ each represent an acidic amino acid residue; and d represents        a hydrophobic amino acid residue.

In one embodiment of the invention, b₃ and b₄ in the amino acid sequenceeach represent a hydrophobic amino acid residue.

In another embodiment of the invention, all of b₁ to b₆ in the aminoacid sequence each represent a hydrophobic amino acid residue.

In still another embodiment of the invention, b₁ to b₆ in the amino acidsequence each independently represent an alanine residue, a valineresidue, a leucine residue, or an isoleucine residue.

In still another embodiment of the invention, d in the amino acidsequence represents an alanine residue, a valine residue, a leucineresidue, or an isoleucine residue.

In still another embodiment of the invention, the self-assemblingpeptide includes a peptide formed of an amino acid sequence ofRLDLRLLLRLDLR (SEQ ID NO: 1), RLDLRLLLRLDLR (SEQ ID NO: 2),RADLRLALRLDLR (SEQ ID NO: 6), RLDLRLALRLDAR (SEQ ID NO: 7),RADLRLLLRLDLR (SEQ ID NO: 8), RADLRLLLRLDAR (SEQ ID NO: 9),RLDLRALLRLDLR (SEQ ID NO: 10), or RLDLRLLARLDLR (SEQ ID NO: 11).

In still another embodiment of the invention, the self-assemblingpeptide includes a peptide formed of an amino acid sequence ofRLDLRLALRLDLR(SEQ ID NO: 1) or RLDLRLLLRLDLR(SEQ ID NO: 2).

According to another aspect of the invention, a modified peptide isprovided. The modified peptide includes the self-assembling peptidewhose N-terminal amino group and/or C-terminal carboxyl group are/ismodified and has a self-assembling ability.

In one embodiment of the invention, the N-terminal amino group and/orthe C-terminal carboxyl group have/has added thereto an amino acidsequence including RGD.

According to still another aspect of the invention, a peptide gel isprovided. The peptide gel is formed from an aqueous solution includingthe self-assembling peptide and/or the modified peptide.

In one embodiment of the invention, the aqueous solution furtherincludes an additive.

In another embodiment of the invention, the additive includes at leastone selected from the group consisting of a pH adjuster, an amino acid,a vitamin, a saccharide, a polysaccharide, an alcohol, a polyalcohol, apigment, a bioactive substance, an enzyme, an antibody, DNA, and RNA.

In still another embodiment of the invention, the peptide gel has anabsolute value L (g/s) of an amount of change in load per unit time of0.03 g/s or more in an approximate straight line of values measured fromthe start of compression to between 8 and 10 seconds in a compressiontest carried out at a compression speed of 0.05 mm/s using a jig whosetip has a spherical shape with a diameter of 3.2 mm and a curvatureradius of 1.6 mm under a temperature condition of 22° C.

According to still another aspect of the invention, a substrate for cellculture is provided. The substrate for cell culture includes at leastone selected from the group consisting of the self-assembling peptide,the modified peptide, and the peptide gel.

According to still another aspect of the invention, a manufacturingmethod for a sterile peptide is provided. The manufacturing method for asterile peptide includes the step of sterilizing the self-assemblingpeptide and/or the modified peptide under a pressurized condition at100° C. or more.

According to still another aspect of the invention, a manufacturingmethod for an article coated with a peptide gel is provided. Themanufacturing method for an article coated with a peptide gel includesthe steps of: freezing the peptide gel; melting the frozen peptide gelto provide a peptide sol; coating at least part of a surface of anarticle to be coated with the peptide sol; and reconstructing a peptidegel from the peptide sol.

Advantageous Effects of Invention

According to the self-assembling peptide having a specific amino acidsequence of the present invention, the peptide gel with practicalmechanical strength may be obtained.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic view for describing a distance between peptideseach formed of a sequence of n-RADARAAARADAR-c. The bold line in a mainchain connecting the N-terminus and the C-terminus in the figurerepresents a peptide bond.

FIG. 2 is a graph showing the results of a compression test on a peptidegel of Example 1.

FIG. 3( a) is a photograph of a peptide gel of Example 1 grasped withtweezers, FIG. 3( b) is a photograph of a peptide gel of Example 2grasped with tweezers, and FIG. 3( c) is a photograph of a peptide gelof Comparative Example 1 grasped with tweezers.

FIG. 4 is a graph showing the results of a compression test on a peptidegel of Example 2.

FIG. 5 is a graph showing the results of a compression test on a peptidegel of Example 3.

FIG. 6 is a graph showing the results of a compression test on a peptidegel of Comparative Example 1.

FIG. 7 is a graph showing the results of a compression test on a peptidegel of Comparative Example 2.

FIG. 8 is a graph showing the results of a compression test on a peptidegel of Example 4.

FIG. 9 is a graph showing cell growth rates in Example 7.

FIG. 10 (a) shows the mass spectrometry results of a peptide aqueoussolution before sterilization treatment, and FIG. 10( b) shows the massspectrometry results of the peptide aqueous solution after sterilizationtreatment.

FIG. 11 (a) shows the mass spectrometry results of a trade name“PuraMatrix™” (manufactured by 3-D Matrix, Ltd.) before sterilizationtreatment, and FIG. 11 (b) shows the mass spectrometry results of thetrade name “PuraMatrix™” (manufactured by 3-D Matrix, Ltd.) aftersterilization treatment.

FIGS. 12( a), 12(b), and 12(c) are a photograph of a gel transferred toa laboratory dish, a photograph of a frozen gel, and a photograph of alaboratory dish whose entire surface is uniformly coated with a peptidegel, respectively.

FIGS. 13( a), 13(b), and 13(c) are a photograph of a gel transferred toa slide glass, a photograph of a frozen gel, and a photograph of a slideglass whose entire surface is uniformly coated with a peptide gel,respectively.

DESCRIPTION OF EMBODIMENTS A. Definition of Terms

(1) The term “self-assembling peptide” as used herein refers to apeptide that assembles spontaneously via an interaction between peptidemolecules in a solvent. Examples of the interaction include, but notparticularly limited to, a hydrogen bond, an interionic interaction, anelectrostatic interaction such as van der Waals force, and a hydrophobicinteraction. In one embodiment, the self-assembling peptides are capableof self-assembling to form nanofibers, which further form a gel in anaqueous solution (for example, a 0.4 w/v % peptide aqueous solution) atroom temperature.(2) The term “gel” as used herein refers to a viscoelastic substancehaving both of viscous property and elastic property.(3) The term “hydrophilic amino acid” as used herein encompasses basicamino acids such as arginine (Arg/R), lysine (Lys/K), and histidine(His/H), acidic amino acids such as aspartic acid (Asp/D) and glutamicacid (Glu/E), and uncharged polar amino acids such as tyrosine (Tyr/Y),serine (Ser/S), threonine (Thr/T), asparagine (Asn/N), glutamine(Gln/Q), and cysteine (Cys/C). The alphabetical letters in parenthesesin the foregoing denote the three-letter code and single-letter code ofthe amino acid, respectively.(4) The term “hydrophobic amino acid” as used herein encompassesnonpolar amino acids such as alanine (Ala/A), leucine (Leu/L),isoleucine (Ile/I), valine (Val/V), methionine (Met/M), phenylalanine(Phe/F), tryptophan (Trp/W), glycine (Gly/G), and proline (Pro/P). Thealphabetical letters in parentheses in the foregoing denote thethree-letter code and single-letter code of the amino acid,respectively.

B. Self-Assembling Peptide

A self-assembling peptide of the present invention is formed of thefollowing amino acid sequence:

-   -   Amino acid sequence: a₁b₁c₁b₂a₂b₃ db₄a₃b₅c₂b₆a₄        where: a₁ to a₄ each represent a basic amino acid residue; b₁ to        b₆ each represent an uncharged polar amino acid residue and/or a        hydrophobic amino acid residue, provided that at least five        thereof each represent a hydrophobic amino acid residue; c₁ and        c₂ each represent an acidic amino acid residue; and d represents        a hydrophobic amino acid residue.

In general, it is estimated that the self-assembling peptide forms aβ-sheet structure having a plane in which a hydrophilic side chain isdisposed and a plane in which a hydrophobic side chain is disposed in anaqueous solution and that a plurality of peptide molecules assemblespontaneously through interactions such as a hydrogen bond or aninterionic interaction acting on hydrophilic planes and a hydrophobicinteraction acting on hydrophobic planes. Therefore, it has beenregarded as very important for a conventional self-assembling peptide tohave a hydrophilic amino acid and a hydrophobic amino acid alternatelyarranged at an equal ratio (see, for example, Patent Document 1).

On the other hand, as described above, the self-assembling peptide ofthe present invention is formed of an amino acid sequence of 13 residueshaving basic amino acid residues (at positions 1, 5, 9, and 13) andacidic amino acid residues (at positions 3 and 11) every other residueat symmetric positions in the N-terminal direction and the C-terminaldirection with respect to a hydrophobic amino acid residue at position 7in the center. That is, one feature of the self-assembling peptide ofthe present invention resides in that the self-assembling peptide doesnot have a hydrophilic amino acid and a hydrophobic amino acidalternately. Further, another feature of the self-assembling peptide ofthe present invention resides in that the self-assembling peptide doesnot have a hydrophilic amino acid residue and a hydrophobic amino acidresidue at an equal ratio. Further, still another feature of theself-assembling peptide of the present invention resides in that theself-assembling peptide has four basic amino acid residues and twoacidic amino acid residues at given symmetric positions with respect toa hydrophobic amino acid residue at position 7 in the center, and bothof amino acid residues at the N-terminus and the C-terminus are basicamino acid residues. The hydrophobic amino acid residue at position 7 isa disadvantage for the formation of a β-sheet structure and makes aratio between a hydrophilic amino acid and a hydrophobic amino acidunequal. Therefore, in general, this has been considered as adverselyaffecting the self-assembling ability of a peptide. However, theself-assembling peptide of the present invention has an excellentself-assembling ability and is capable of forming a peptide gel moreexcellent in mechanical strength than ever before. Although the reasonwhy such effect is exerted is not clear, a possible reason is asdescribed below. The self-assembling peptide of the present inventionnot only has a hydrophobic amino acid at position 7 but also has basicamino acid residues and acidic amino acid residues in which the numberof the basic amino acid residues is larger than the number of the acidicamino acid residues by two and has the respective amino acid residues atspecific positions. As a result, an electrostatic attractive force andan electrostatic repulsive force act on peptide molecules at anextremely excellent balance while an ability to form a β-sheet structureis maintained.

An amino acid that constructs the self-assembling peptide may be anL-amino acid or a D-amino acid. Further, the amino acid may be a naturalamino acid or a non-natural amino acid. Of those, a natural amino acidis preferred because it is available at a low price and facilitatespeptide synthesis.

In the amino acid sequence, a₁ to a₄ each represent a basic amino acidresidue. A basic amino acid is preferably arginine, lysine, orhistidine, more preferably arginine or lysine. This is because thoseamino acids have strong basicity. a₁ to a₄ may represent the same aminoacid residue or different amino acid residues.

In the amino acid sequence, b₁ to b₆ each represent an uncharged polaramino acid residue and/or a hydrophobic amino acid residue, and at leastfive thereof each represent a hydrophobic amino acid residue. Ahydrophobic amino acid is preferably alanine, leucine, isoleucine,valine, methionine, phenylalanine, tryptophan, glycine, or proline. Anuncharged polar amino acid is preferably tyrosine, serine, threonine,asparagine, glutamine, or cysteine. This is because those amino acidsare easily available.

b₃ and b₄ each independently represent preferably any appropriatehydrophobic amino acid residue, more preferably a leucine residue, analanine residue, a valine residue, or an isoleucine residue,particularly preferably a leucine residue or an alanine residue. In theamino acid sequence, when b₃ and b₄, which are located at positions 6and 8, respectively, each represent a hydrophobic amino acid residue,three amino acid residues at positions 6 to 8 are consecutivelyhydrophobic amino acid residues. It is estimated that a hydrophobicregion formed in the center of the amino acid sequence as describedabove is capable of contributing to the formation of a peptide gelexcellent in strength through its hydrophobic interaction and the like.

All of b₁ to b₆ each preferably represent a hydrophobic amino acidresidue. This is because the self-assembling peptide is capable ofsuitably forming a β-sheet structure and self-assembling. b₁ to b₆ eachindependently represent more preferably a leucine residue, an alanineresidue, a valine residue, or an isoleucine residue, still morepreferably a leucine residue or an alanine residue. In a preferredembodiment, four or more of b₁ to b₆ each represent a leucine residue,it is particularly preferred that five or more thereof each represent aleucine residue, and it is most preferred that all thereof eachrepresent a leucine residue. This is because a self-assembling peptidethat is excellent in water solubility and is capable of forming ahigh-strength peptide gel may be obtained.

In the amino acid sequence, c₁ and c₂ each represent an acidic aminoacid residue. An acidic amino acid is preferably aspartic acid orglutamic acid. This is because those amino acids are easily available.c₁ and c₂ may be the same amino acid residue or different amino acidresidues.

In the amino acid sequence, d represents a hydrophobic amino acidresidue. As described above, the self-assembling peptide has ahydrophobic amino acid residue as d and a given symmetric structure,thereby it is conceivable that the self-assembling peptide is capable offorming a gel more excellent in mechanical strength than a conventionalpeptide gel. Although the reason why such effect is exerted is notunclear, a possible reason is as described below. That is, it isestimated that the self-assembling peptide of the present invention iscapable of forming an assembled state having high uniformity because theamino acid residue d at position 7 is a hydrophobic amino acid residue,which makes an overlap of peptide molecules constant in self-assembling.

d preferably represents an alanine residue, a valine residue, a leucineresidue, or an isoleucine residue. In this case, amino acids on thehydrophilic plane side of a β-sheet structure formed by theself-assembling peptide may be non-complementary in side chain length toeach other. However, the self-assembling peptide is capable ofexhibiting an excellent self-assembling ability and is capable offorming a peptide gel excellent in mechanical strength than ever before.This is an effect different greatly from the conventional finding thatamino acids on the hydrophilic plane side of a β-sheet structure arepreferably complementary in side chain length to each other in order toobtain an electrostatic attractive force suitable for self-assembling.The term “complementary in side chain length” as used herein means thatthe sum of the numbers of atoms mainly involved in side chain lengths ofa pair of amino acid residues (for example, a basic amino acid residueand an acidic amino acid residue) which exhibits an interaction isconstant. For example, FIG. 1 is a schematic view for describing adistance between peptide molecules having amino acid residuesnon-complementary in side chain length to each other. As shown in FIG.1, the sum (7) of the numbers of atoms which are mainly involved in sidechain lengths of an alanine residue-arginine residue pair surrounded bya dotted line is smaller than the sum (9) of the numbers of atoms whichare mainly involved in side chain lengths of an aspartic acidresidue-arginine residue pair surrounded by a solid line.

The total sum of charges in a neutral pH environment of amino acidresidues included in the self-assembling peptide is substantially +2.That is, in the self-assembling peptide, a positive charge and anegative charge derived from side chains of amino acid residues includedin the peptide in a neutral pH environment are not cancelled. Inaddition, both of amino acid residues at the N-terminus and theC-terminus are basic amino acid residues. Thus, in the self-assemblingpeptide of the present invention, for example, not only an electrostaticattractive force but also an electrostatic repulsive force act onpeptide molecules, a delicate balance between those forces ismaintained, and hence the self-assembling peptide is substantially freeof excessive association. Accordingly, it is estimated that theself-assembling peptide is capable of forming a stable gel without beingprecipitated in a neutral pH environment. It should be noted that theterm “neutral pH environment” as used herein refers to a region having apH of 6 to 8, preferably a pH of 6.5 to 7.5.

The charges of the self-assembling peptide at each pH may be counted inaccordance with, for example, the Lehninger (Biochimie, 1979) method.The Lehninger method may be conducted with a usable program found, forexample, on the website of EMBL WWW Gateway to Isoelectric Point Service(http://www.embl-heidelberg.de/cgi/pi-wrapper.pl).

An aqueous solution including the self-assembling peptide is capable offorming a peptide gel excellent in mechanical strength. In oneembodiment, the self-assembling peptide aqueous solution (preferably 0.2to 5 w/v %, more preferably 0.2 to 2 w/v %, particularly preferably 0.2to 1 w/v %, most preferably 0.3 to 0.8 w/v %) is capable of forming agel having an absolute value L (g/s) of an amount of change in load perunit time in an approximate straight line of values measured at theinitial stage (from the start of compression to between 8 and 10seconds) after the start of compression of preferably 0.03 g/s or more,more preferably 0.035 g/s or more, particularly preferably 0.04 g/s ormore in a compression test carried out at a compression speed of 0.05mm/s using a jig whose tip has a spherical shape with a diameter of 3.2mm and a curvature radius of 1.6 mm under a temperature condition of 22°C. As described in examples later, the compression test may be carriedout, for example, by using a viscoelasticity measurement apparatus(product number “RSA III” manufactured by TA Instruments) equipped witha jig made of stainless-steel (manufactured by TA Instruments).

A self-assembling peptide according to a preferred embodiment of thepresent invention is exemplified below.

(SEQ ID NO: 1) n-RLDLRLALRLDLR-c (SEQ ID NO: 2) n-RLDLRLLLRLDLR-c(SEQ ID NO: 6) n-RADLRLALRLDLR-c (SEQ ID NO: 7) n-RLDLRLALRLDAR-c(SEQ ID NO: 8) n-RADLRLLLRLDLR-c (SEQ ID NO: 9) n-RADLRLLLRLDAR-c(SEQ ID NO: 10) n-RLDLRALLRLDLR-c (SEQ ID NO: 11) n-RLDLRLLARLDLR-c

The self-assembling peptide may be manufactured by any appropriatemanufacturing method. Examples thereof include a chemical synthesismethod such as a solid-phase method such as an Fmoc method or aliquid-phase method and a molecular biological method such as generecombinant expression.

C. Modified Peptide

The modified peptide of the present invention includes theabove-mentioned self-assembling peptide subjected to any appropriatemodification as long as the self-assembling ability is maintained. Asite at which the modification is carried out may be an N-terminal aminogroup of the self-assembling peptide, may be a C-terminal carboxyl groupthereof, or may be both of the groups.

Any appropriate modification may be selected as the modification in sucha range that the resultant modified peptide has a self-assemblingability. Examples of the modification include: introduction of aprotective group such as acetylation of an N-terminal amino group oramidation of a C-terminal carboxyl group; introduction of a functionalgroup such as alkylation, esterification, or halogenation;hydrogenation; introduction of a saccharide compound such as amonosaccharide, a disaccharide, an oligosaccharide, or a polysaccharide;introduction of a lipid compound such as a fatty acid, a phospholipid,or a glycolipid; introduction of an amino acid or a protein;introduction of DNA; and introduction of any other compound having abioactivity or the like. When the amino acid or the protein isintroduced, the peptide after the introduction is an added peptide inwhich any appropriate amino acid is added to the self-assembling peptideat the N-terminus and/or the C-terminus. In this description, the addedpeptide is also included in the modified peptide. The modifications maybe performed alone or in combination. For example, the added peptide inwhich a desired amino acid has been introduced into the above-mentionedself-assembling peptide at the C-terminus may be acetylated at theN-terminus and amidated at the C-terminus.

The added peptide (modified peptide) does not have features of theabove-mentioned self-assembling peptide as a whole in some cases.Specific examples thereof include a case where the addition of anyappropriate amino acid makes a sequence in the N-terminal direction anda sequence in the C-terminal direction asymmetric with respect to ahydrophobic amino acid residue at position 7 in the center, and a casewhere a hydrophobic amino acid residue and a hydrophilic amino acidresidue are included at an equal ratio. Even in such case, theself-assembling peptide has an extremely excellent self-assemblingability, and hence the added peptide obtained by adding any appropriateamino acid to the self-assembling peptide is also capable of forming apeptide gel excellent in mechanical strength.

When the amino acid or the protein is introduced, the number of aminoacid residues that construct the modified peptide after the introductionis preferably 14 to 200, more preferably 14 to 100, more preferably 14to 50, particularly preferably 14 to 30, most preferably 14 to 20. Thisis because when the number of amino acid residues exceeds 200, theself-assembling ability of the self-assembling peptide may be impaired.

The kind and position of the amino acid to be introduced may beappropriately set depending on applications of the modified peptide andthe like. It is preferred that a hydrophobic amino acid and ahydrophilic amino acid be introduced alternately from an arginineresidue (hydrophilic amino acid) at the N-terminus and/or the C-terminusof the self-assembling peptide.

Specific examples of the amino acid to be introduced include: REDV,EILDV, YEKPGSPPREVVPRPRPGV, KNNOKSEPLIGRK, YIGSR, RNIAELLKDI,RYVVLPRPVCFEKGMNYTVR, IKVAV, PDSGR, amino acid sequences including RGDsequences (for example, GRGDSPASS, RGDN, RGDF, RGDT, RGDA, RGD, andRGDS), and the like as cell adhesion factors; PPKKKRKV, PAAKRVKLD,PQPKKKP, QRKRQK, and the like as nuclear transport signals;MMSFVSLLLVGILFWATEAEQLTLCEVFQ and the like as endoplasmic reticulumtransport signals; and MLSLRQSIRFFLPATRTLCSSRYLL and the like asmitochondrial transport signals. Those sequences may be introduced aloneor in combination. Further, the amino acid sequence to be introduced andthe self-assembling peptide may be linked together via one or more ofany appropriate amino acids.

The modification may be performed by any appropriate method depending onthe kinds and the like.

An aqueous solution including the modified peptide is capable of forminga peptide gel excellent in mechanical strength. In one embodiment, amodified peptide aqueous solution (preferably 0.2 to 5 w/v %, morepreferably 0.2 to 2 w/v %, particularly preferably 0.2 to 1 w/v %, mostpreferably 0.3 to 0.8 w/v %) is capable of forming a gel having anabsolute value L (g/s) of an amount of change in load per unit time inan approximate straight line of values measured at the initial stage(from the start of compression to between 8 and 10 seconds) after thestart of compression of preferably 0.03 g/s or more, more preferably0.035 g/s or more, particularly preferably 0.04 g/s or more in acompression test carried out at a compression speed of 0.05 mm/s using ajig whose tip has a spherical shape with a diameter of 3.2 mm and acurvature radius of 1.6 mm under a temperature condition of 22° C.

D. Peptide Gel

The peptide gel of the present invention is formed from an aqueoussolution including the self-assembling peptide and/or the modifiedpeptide (hereinafter, “the self-assembling peptide and/or the modifiedpeptide” are/is sometimes referred to as “the peptide of the presentinvention”). It is estimated that the peptide of the present inventionassembles spontaneously in an aqueous solution, forms a fiber-likemolecule assembly having a width at a nanometer scale, the so-callednanofibers, and forms a three dimensional network structure mainly owingto an electrostatic interaction acting on the nanofibers, and then formsa gel. The aqueous solution may include only one kind or two or morekinds of the peptide of the present invention. The aqueous solution mayfurther include any appropriate additive in addition to the peptide ofthe present invention and water. Further, the aqueous solution mayinclude insoluble matter such as a cell.

The concentration of the peptide of the present invention in the aqueoussolution is preferably 0.2 to 5 w/v %, more preferably 0.2 to 2 w/v %,particularly preferably 0.2 to 1 w/v %, most preferably 0.3 to 0.8 w/v%. When the concentration falls within such a range, a peptide gelexcellent in mechanical strength may be obtained. Further, when thepeptide gel is used as a substrate for cell culture, a satisfactory cellsurvival rate may be provided.

The additive may be appropriately selected depending on applications ofthe peptide gel, kinds of the peptide included therein, and the like.Specific examples of the additive include: pH adjusters such as sodiumhydroxide, potassium hydroxide, hydrochloric acid, phosphoric acid,sodium hydrogen carbonate, and sodium carbonate; amino acids; vitaminssuch as vitamin A, vitamin B family, vitamin C, vitamin D, vitamin E,and derivatives thereof; saccharides such as a monosaccharide, adisaccharide, and an oligosaccharide; polysaccharides such as hyaluronicacid, chitosan, and hydrophilic cellulose; alcohols such as ethanol,propanol, and isopropanol; polyhydric alcohols such as glycerin andpropylene glycol; pigments such as phenol red; bioactive substances suchas hormones, cytokines (including hemopoietic factors and growthfactors), and peptides; enzymes; antibodies; DNA; RNA; and other generallow-molecular compounds. The additives may be added alone or incombination. The concentration of the additive in the aqueous solutionmay be appropriately set depending on the purposes, applications of thepeptide gel, and the like.

Specific examples of the aqueous solution including the additive includevarious buffers such as phosphate buffered saline (PBS) and Tris-HCl, amedium for cell culture such as a Dulbecco's modified Eagle medium(DMEM), and an aqueous solution having a pH adjusted with sodiumhydroxide, hydrochloric acid, sodium hydrogen carbonate, or the like.

The aqueous solution may have any appropriate pH depending on thepurposes. For example, the aqueous solutions before and after dissolvingthe peptide of the present invention each have a pH of preferably 5 to9, more preferably 5.5 to 8, particularly preferably 6.0 to 7.5. Whenthe pH falls within such a range, a peptide gel excellent in mechanicalstrength may be obtained. In addition, a satisfactory cell survival ratemay be obtained when the aqueous solution includes a cell. Further, whenthe pH falls within such a range, peptide degradation hardly occursunder a high-temperature and pressurized condition, and hence thepeptide gel may be subjected to high-pressure steam sterilizationtreatment such as autoclaving. As a result, a peptide gel in a sterilestate may be obtained in a simple manner.

Any appropriate cell may be selected as the cell depending on thepurposes and the like. The cell may be an animal cell or a plant cell.Specific examples of the cell include cartilage cells, myoblasts, bonemarrow cells, fibroblasts, hepatocytes, and cardiomyocytes.

The peptide gel of the present invention has an absolute value L (g/s)of an amount of change in load per unit time of preferably 0.03 g/s ormore, more preferably 0.035 g/s or more, particularly preferably 0.04g/s or more in an approximate straight line of values measured at theinitial stage (from the start of compression to between 8 and 10seconds) after the start of compression in a compression test carriedout at a compression speed of 0.05 mm/s using a jig whose tip has aspherical shape with a diameter of 3.2 mm and a curvature radius of 1.6mm under a temperature condition of 22° C. The peptide gel with suchmechanical strength may be formed, for example, from an aqueous solutionincluding the peptide of the present invention at a concentration ofpreferably 0.2 to 5 w/v %, more preferably 0.2 to 2 w/v %, particularlypreferably 0.2 to 1 w/v %, most preferably 0.3 to 0.8 w/v %.

The peptide gel of the present invention has a visible lighttransmittance, which is measured at an absorbance of 380 nm to 780 nm ina cell having an optical path length of 10 mm, of preferably 50% ormore, more preferably 70% or more, particularly preferably 90% or more.The peptide gel having such visible light transmittance may be formed,for example, from an aqueous solution including the peptide of thepresent invention at concentration of 0.2 to 2 w/v %. Further, areduction rate in visible light transmittance (%) after the peptide gelhas been left to stand in a sealed state at room temperature for a longperiod of time (for example, 2 months) (100−(visible light transmittanceafter storage/visible light transmittance before storage×100)) ispreferably 30% or less, more preferably 20% or less, particularlypreferably 10% or less. The peptide gel having such high visible lighttransmittance has advantages such as facilitating cell observation witha fluorescent light microscope or the like when used as a substrate forcell culture. The visible light transmittance may be measured, forexample, by using a UV/VIS measurement apparatus.

The peptide gel may be formed by any appropriate method. Typically, thepeptide gel may be formed by leaving an aqueous solution including atleast one kind of the peptide of the present invention to stand still.The temperature or time in leaving the aqueous solution to stand stillis not particularly limited as long as the peptide of the presentinvention self-assembles to form a gel, and may be appropriately setdepending on use purposes of the gel, kinds of the peptide,concentrations, and the like. The time for leaving the aqueous solutionto stand still is generally 1 minute or more, preferably 3 minutes ormore, more preferably 5 minutes or more. The temperature is generally 4to 50° C., preferably 15 to 45° C.

E. Applications of Self-Assembling Peptide, Modified Peptide, andPeptide Gel

Preferred applications of the self-assembling peptide, modified peptide,and peptide gel of the present invention include, for example:substrates for cell culture; cosmetics such as skin care products andhair care products; medical products such as decubitus preparations,bone fillers, injectable agents for aesthetic, adjuvant to ophthalmicoperation, artificial vitreous bodies, artificial lenses, jointlubricants, ophthalmic solutions, DDS substrates, and hemostats; waterretention materials for moistening; desiccants; and coating agents formedical devices such as contact lenses.

F. Substrate for Cell Culture

The substrate for cell culture of the present invention includes atleast one selected from the self-assembling peptide, the modifiedpeptide, and the peptide gel described above. The substrate for cellculture of the present invention is formed from a self-assemblingpeptide and/or a modified peptide obtained by chemical synthesis. Hence,there is no risk of contamination with a pathogen and the like and cellculture may be carried out safely. Further, the gel formed from thepeptide of the present invention may be transparent in a neutral pHenvironment and excellent in mechanical strength, and hence thesubstrate for cell culture of the present invention is excellentinvisibility and handleability during cell culture.

In the interior of the substrate for cell culture, the peptides of thepresent invention self-assemble, form into fibers, and form a threedimensional network structure. Thus, not only culture on the substratefor cell culture but also culture in the substrate for cell culture maybe carried out.

When culture is carried out on the substrate for cell culture, cells tobe cultured may be placed and cultured on the preformed peptide gelincluding the peptide of the present invention. When culture is carriedout in the substrate for cell culture, the peptide of the presentinvention or the peptide aqueous solution is mixed with cells or a cellsuspension, and a peptide gel may be formed from the mixture to culturethe cells.

A liquid phase of the peptide gel may be replaced by a desired culturesolution through solvent replacement. The solvent replacement may becarried out, for example, by using a trade name “Cell Culture Insert” orthe like. The details about the peptide gel (for example, peptideconcentration, kind of an additive which may be included in an aqueoussolution (mixture), and pH) and a forming method therefor are asdescribed in the section D.

Any appropriate cell may be selected as the cell to be cultureddepending on the purposes and the like. The cell may be an animal cellor a plant cell. Specific examples of the cell include cartilage cells,myoblasts, bone marrow cells, fibroblasts, hepatocytes, andcardiomyocytes. A culture solution and a culture condition may beappropriately selected depending on kinds of cells to be cultured,purposes, and the like.

The substrate for cell culture of the present invention is excellent inbiocompatibility and safety, and hence may be suitably utilized, forexample, in three dimensional cell culture in the regenerative medicinefield or the like.

G. Manufacturing Method for Sterile Peptide

A manufacturing method for a sterile peptide of the present inventionincludes the step of sterilizing the self-assembling peptide and/or themodified peptide under a pressurized condition at 100° C. or more. Thosepeptides are typically subjected to sterilization treatment in the formof a peptide aqueous solution or a peptide gel formed from the peptideaqueous solution. The peptide aqueous solution has a pH of preferably 5to 9, more preferably 5.5 to 8, particularly preferably 6.0 to 7.5. Whenthe peptide aqueous solution has such pH, peptide degradation does notsubstantially occur even in sterilization under a temperature conditionof 100° C. or more, and hence the peptide of the present invention in asterile state may be obtained. The peptide aqueous solution and thepeptide gel are as described in the section D.

Any appropriate sterilization method may be adopted as the sterilizationmethod. For example, a high-temperature and high-pressure saturatedwater vapor sterilization (so-called autoclave sterilization) method maybe preferably employed. The pressure during the autoclave sterilizationis preferably 0.122 to 0.255 MPa, more preferably 0.152 to 0.233 MPa.Further, the sterilization temperature is preferably 105 to 135° C.,more preferably 110 to 125° C. Further, the sterilization time ispreferably 1 to 60 minutes, more preferably 3 to 40 minutes,particularly preferably 5 to 30 minutes.

The autoclave sterilization may be performed using a commerciallyavailable autoclave apparatus.

H. Manufacturing Method for Article Coated with Peptide Gel

A manufacturing method for an article coated with a peptide gel of thepresent invention includes the steps of: freezing the peptide gel(freezing step); melting the frozen peptide gel to provide a peptide sol(melting step); coating at least part of a surface of an article to becoated with the peptide sol (coating step); and reconstructing a peptidegel from the peptide sol (gelling step). The method may further includeany appropriate step as necessary. When the peptide gel of the presentinvention is frozen and melted, a bond between peptide molecules iscleaved, and hence a three dimensional network structure that constructsthe gel collapses. Thus, a sol having peptide molecules uniformlydispersed in an aqueous solution may be obtained. At least part of thesurface of an article to be coated is coated with the sol with highuniformity and the sol is then turned into a gel. Thus, the surface ofthe article may be uniformly coated with the peptide gel.

H-1. Freezing Step

Any appropriate condition may be adopted as a freezing condition as longas the peptide gel is frozen. A freezing temperature has only to beequal to or less than a temperature at which the peptide gel is frozen.A freezing speed is also not limited, and the peptide gel may begradually frozen or quickly frozen. For example, the peptide gel may besuitably frozen by placing the peptide gel under a temperature conditionof −10° C. or less.

Any appropriate freezing means may be selected as freezing means, suchas a household or industrial freezer or liquid nitrogen. It should benoted that the frozen peptide gel may be preserved in a frozen state forany appropriate period of time before the melting step.

When the peptide gel to be frozen is a gel formed from a peptide aqueoussolution including an additive, the concentration of the additive ispreferably such a concentration that does not adversely affect gelreconstruction in the gelling step. The concentration may beappropriately set depending on the kinds, concentrations, and the likeof the peptide. In general, however, a low concentration is preferred.For example, in the case of HEPES and a Tris-HCl solution, the finalconcentration is preferably 50 mM or less, more preferably 40 mM orless. In the case of a sodium hydrogen carbonate solution and a sodiumcarbonate solution, the final concentration is preferably 5 mM or less,more preferably 4 mM or less. In the case of a PBS solution, the finalconcentration is preferably 0.5×PBS or less, more preferably 0.3×PBS orless. Further, in the case of Pharmacopoeia physiological saline, thefinal concentration is preferably 0.5 wt % or less, more preferably 0.4wt % or less.

H-2. Melting Step

A melting temperature may be set to any appropriate temperature as longas it is a temperature at which the frozen peptide gel obtained in thefreezing step is melted to form a sol. The frozen peptide gel may bemelted at a constant temperature, or may be melted at differenttemperatures in a stepwise manner. A melting speed and time is notlimited, and the frozen peptide gel may be gradually melted or quicklymelted. The frozen peptide gel may suitably melted, for example, byplacing the frozen peptide gel under a temperature condition of 5 to 70°C., preferably 15 to 45° C.

Any appropriate means may be selected as melting means. Specificexamples of the melting means include a water bath, an oil bath, and athermostat bath.

When the peptide gel is frozen and melted as described above, a varietyof bonds between peptide molecules that form the gel are cleaved toyield a sol. The sol obtained by freezing and melting has a remarkablyreduced viscosity because a variety of bonds between peptide moleculeshave been sufficiently cleaved, and hence is capable of uniformlycoating the surface of an article to be coated without difficulty. Itshould be noted that, from the viewpoint of providing higher uniformityof the sol, the frozen peptide gel may be melted while vibrated to suchan extent that air bubbles are not generated in the resultant sol, orthe sol obtained by melting may be vibrated and then subjected to thecoating step. Examples of such vibration method include shaking thefrozen peptide gel or sol and applying ultrasound thereto.

H-3. Coating Step

Any appropriate method may be adopted as a coating method. Specificexamples thereof include a dispenser coating mode, an immersion mode, abar coater mode, a mode including applying a sol onto the surface of anarticle to be coated with a centrifugal force, and a mode includingapplying a sol fluidized by tilting an article to be coated onto thesurface thereof. In the sol, a variety of bonds between peptidemolecules are sufficiently cleaved to reduce its viscosity remarkably,and peptide molecules are sufficiently dispersed. Thus, the sol iscapable of forming a uniform layer on the surface of an article to becoated.

Any appropriate article may be adopted as the article to be coated.Examples thereof include a container such as a tube or a bottle, a cellculture instrument such as a multi-well dish or a laboratory dish, and aplate such as a slide glass. The article to be coated is formed of anyappropriate material such as glass, plastic, or metal.

H-4. Gelling Step

A Condition for reconstructing a gel (temperature, time, or the like) isnot limited as long as a peptide gel is reconstructed, and may beappropriately set depending on kinds, concentrations, and the like ofthe peptide. The peptide of the present invention has a self-assemblingability and hence is capable of self-assembling and reconstructing a gelspontaneously by setting the condition to an appropriate condition.

As the condition for reconstructing a gel, for example, it is sufficientto reconstruct a gel that an article whose surface is coated with thepeptide sol is left to stand still. A temperature in leaving the articleto stand still is preferably 15° C. or more, more preferably 25 to 45°C. A time in leaving the article to stand still is preferably 1 minuteor more, more preferably 5 minutes or more.

The thickness of the peptide gel reconstructed in the gelling step, thatis, the thickness of a coating film on an article surface may be, forexample, 1 μm or more, preferably 1 μm to 1 cm.

EXAMPLES

Hereinafter, the present invention is specifically described by way ofexamples. However, the present invention is by no means limited by theseexamples.

Example 1

A self-assembling peptide formed of an amino acid sequence of SEQ ID NO:1 described in Table 1 was synthesized by an Fmoc solid-phase synthesismethod. Next, the self-assembling peptide was acetylated at theN-terminus and amidated at the C-terminus by a conventional method toafford a modified peptide 1 ([CH₃CO]-RLDLRLALRLDLR-[NH₂]).

The resultant modified peptide 1 was dissolved in a 0.1 wt % sodiumhydrogen carbonate solution at concentrations of 0.2, 0.4, and 0.6 w/v %to afford peptide solutions. The resultant peptide solutions were eachmeasured for a pH value using pH test paper (trade name: pH IndicatorPapers, manufactured by Whatman International Ltd., measurement rangepH=6.0 to 8.1, Cat. No. 2629 990), and the pH was found to fall withinthe range of 6.9 to 7.8. 300 μl each of the peptide solutions werecharged into a trade name “Nunc Tissue Culture Inserts” (membranediameter: 10 mm, pore size: 8.0 μm, membrane material: polycarbonate)(product number “Cat. No: 136862” manufactured by Nalge NuncInternational) and left to stand still at 22° C. for 2 hours to formgels. The gels each had a thickness of approximately 2 mm. The resultantgels were subjected to solvent replacement with DMEM for 12 hours toafford peptide gels 1 (liquid phase: DMEM) at peptide concentrations of0.2, 0.4, and 0.6 w/v %. The resultant peptide gels 1 at the respectiveconcentrations were subjected to the following compression test tomeasure mechanical strength.

[Compression Test]

The mechanical strength was measured by compressing each gel at a speedof 0.05 mm/s (s=second) using a viscoelasticity measurement apparatus(product number “RSA III” manufactured by TA Instruments) equipped witha jig made of stainless-steel whose tip has a spherical shape (diameter:3.2 mm, curvature radius: 1.6 mm) (manufactured by TA Instruments) undera condition of 22° C.

FIG. 2 shows the results of the compression test. FIG. 2 shows arelationship between a load applied to the apparatus in graduallycompressing a sample with the jig and a time, and a larger slope of anapproximate straight line indicates higher mechanical strength. Thus,when the absolute value of a slope of an approximate straight line ofvalues measured at the initial stage (from the start of compression tobetween 8 and 10 seconds) after the start of compression (amount ofchange in load per unit time) is defined as L, a larger L valueindicates higher mechanical strength. As shown in FIG. 2, the peptidegel 1 at 0.4 w/v % had an absolute value L of an amount of change inload per unit time of 0.0476 g/s, which was determined for about 10seconds after the start of compression in the compression test.

The peptide gel 1 at 0.4 w/v % was grasped with tweezers andtransported. As a result, as shown in FIG. 3( a), the peptide gel 1 hadsufficient strength to grasp and was excellent in handleability.

Example 2

A modified peptide 2 ([CH₃O]-RLDLRLLLRLDLR-[NH₂]) was obtained in thesame manner as in Example 1 except that an amino acid sequence of SEQ IDNO: 2 was adopted in place of the amino acid sequence of SEQ ID NO: 1.Peptide gels 2 (liquid phase: DMEM) at peptide concentrations of 0.2,0.4, and 0.6 w/v % were formed in the same manner as in Example 1 exceptthat the modified peptide 2 was used in place of the modified peptide 1.

The resultant peptide gels were each measured for mechanical strength inthe same manner as in Example 1. FIG. 4 shows the results. As shown inFIG. 4, the peptide gel 2 at 0.4 w/v % had an absolute value L of anamount of change in load per unit time of 0.0423 g/s in the compressiontest.

The peptide gel 2 at 0.4 w/v % was grasped with tweezers andtransported. As a result, as shown in FIG. 3( b), the peptide gel 2 hadsufficient strength to grasp and was excellent in handleability.

Example 3

A modified peptide 3 ([CH₃CO]-RLDLRLALRLDLRL-[NH₂]) was obtained in thesame manner as in Example 1 except that an amino acid sequence of SEQ IDNO: 3 was adopted in place of the amino acid sequence of SEQ ID NO: 1.Peptide gels 3 (liquid phase: DMEM) at peptide concentrations of 0.2 and0.4 w/v % were formed in the same manner as in Example 1 except that themodified peptide 3 was used in place of the modified peptide 1.

The resultant peptide gels were each measured for mechanical strength inthe same manner as in Example 1. FIG. 5 shows the results. As shown inFIG. 5, the peptide gel 3 at 0.4 w/v % had an absolute value L of anamount of change in load per unit time of 0.0336 g/s in the compressiontest.

Comparative Example 1

A modified peptide c1 ([CH₃CO]-RASARADARADARASA-[NH₂]) was obtained inthe same manner as in Example 1 except that an amino acid sequence ofSEQ ID NO: 4 was adopted in place of the amino acid sequence of SEQ IDNO: 1. Peptide gels c1 (liquid phase: DMEM) at peptide concentrations of0.2, 0.4, and 0.6 w/v % were formed in the same manner as in Example 1except that the modified peptide c1 was used in place of the modifiedpeptide 1.

The resultant peptide gels were each measured for mechanical strength inthe same manner as in Example 1. FIG. 6 shows the results. As shown inFIG. 6, the peptide gel c1 at 0.4 w/v % had an absolute value L of anamount of change in load per unit time of 0.0143 g/s in the compressiontest.

The peptide gel c1 at 0.4 w/v % was grasped with tweezers andtransported. As a result, as shown in FIG. 3( c), the peptide gel c1 hadinsufficient mechanical strength and was problematic in handleability.

Comparative Example 2

A modified peptide c2 ([CH₃CO]-RASARADARASARADA-[NH₂]) was obtained inthe same manner as in Example 1 except that an amino acid sequence ofSEQ ID NO: 5 was adopted in place of the amino acid sequence of SEQ IDNO: 1. Peptide gels c2 (liquid phase: DMEM) at peptide concentrations of0.2 and 0.4 w/v % were formed in the same manner as in Example 1 exceptthat the modified peptide c2 was used in place of the modified peptide1.

The resultant peptide gels were each measured for mechanical strength inthe same manner as in Example 1. FIG. 7 shows the results. As shown inFIG. 7, the peptide gel c2 at 0.4 w/v % had an absolute value L of anamount of change in load per unit time of 0.0167 g/s in the compressiontest.

Example 4

A modified peptide 4 ([CH₃CO]-RGDNRLDLRLALRLDLR-[NH₂]) was obtained inthe same manner as in Example 1 except that an amino acid sequence ofSEQ ID NO: 12 was adopted in place of the amino acid sequence of SEQ IDNO: 1. Peptide gels 4 (liquid phase: DMEM) at peptide concentrations of0.2, 0.4, and 0.6 w/v % were formed in the same manner as in Example 1except that the modified peptide 4 was used in place of the modifiedpeptide 1.

The resultant peptide gels were each measured for mechanical strength inthe same manner as in Example 1. FIG. 8 shows the results. As shown inFIG. 8, the peptide gel 4 at 0.4 w/v % had an absolute value L of anamount of change in load per unit time of 0.0618 g/s in the compressiontest.

TABLE 1 Amino acid sequence Example 1 n-RLDLRLALRLDLR-c SEQ ID No: 1Example 2 n-RLDLRLLLRLDLR-c SEQ ID No: 2 Example 3 n-RLDLRLALRLDLRL-cSEQ ID No: 3 Example 4 n-RGDNRLDLRLALRLDLR-c SEQ ID No: 12 Comparativen-RASARADARADARASA-c SEQ ID No: 4 Example 1 Comparativen-RASARADARASARADA-c SEQ ID No: 5 Example 2

As shown in FIGS. 2 and 4 to 8, it is understood that the peptide of thepresent invention is capable of forming a peptide gel with highmechanical strength as compared to the self-assembling peptide of eachof the comparative examples. Further, as shown in FIG. 3, it isunderstood that the peptide gel of the present invention has highmechanical strength and hence is extremely excellent in handleability.In addition, the peptide of the present invention is capable of forminga peptide gel with sufficient mechanical strength at a low peptideconcentration and hence is advantageous in terms of cost as well.

Example 5

A cell suspension including mouse myoblasts (L6) at a cell concentrationof 2.0×10⁶ cells/ml and a peptide aqueous solution including themodified peptide 1 at 1.0 w/v % were mixed with each other at a volumeratio of 3:2 (cell suspension:peptide aqueous solution). The resultantmixture (cell concentration: 1.2×10⁶ cells/ml, peptide concentration:0.4 w/v %) was charged into a Cell Culture Insert (product number“353096” manufactured by BD Falcon) and left to stand still at roomtemperature for approximately 1 minute to form a peptide gel. The gel inthe Cell Culture Insert was directly set in wells of a 24-well plate fortissue culture (product number “3820-024” manufactured by AGC TECHNOGLASS CO., LTD.) with 1 mL of a DMEM medium containing 10% calf serum.Next, cell culture was carried out in an incubator at 37° C. in thepresence of 5% CO₂. Medium exchange was carried out only once on Day 2after the start of culture. The gel was collected on Days 1, 2, and 4after the start of culture, and DNA quantification was carried out usinga trade name “CyQUANT (registered trademark) Cell Proliferation AssayKit*for cells in culture**1000 assays*” (product number “C7026”manufactured by Invitrogen), to thereby calculate cell growth rates. Thecell growth rates on Days 1, 2, and 4 after the start of culture were150%, 180%, and 310%, respectively, when the cell growth rateimmediately after the start of culture was defined as 100%, that is, thenumber of cells increased in accordance with a lapse of days (an averageof n=3 was used for the calculation results).

Example 6

Cell culture and DNA quantification were carried out in the same manneras in Example 5 except that the modified peptide 2 was used in place ofthe modified peptide 1. As a result of calculation of cell growth rates,the cell growth rates on Days 1, 2, and 4 after the start of culturewere 140%, 160%, and 250%, respectively, when the cell growth rateimmediately after the start of culture was defined as 100%, that is, thenumber of cells increased in accordance with a lapse of days (an averageof n=3 was used for the calculation results).

Example 7

The modified peptide 1 was dissolved in a sodium carbonate solution toprepare a 0.5 w/v % peptide aqueous solution (final concentration ofsodium carbonate: 2.75 mM). The peptide aqueous solution was subjectedto sterilization treatment at 121° C. for 20 minutes using an autoclaveapparatus (product number “MLS 3020” manufactured by SANYO Electric Co.,Ltd.) to afford a peptide gel. The gel and a cell suspension obtained bysuspending mouse NIH3T3 cells in a DMEM medium were uniformly mixed witheach other at a volume ratio of 2:1 (gel:cell suspension) by pipetting.100 μL of the resultant cell-gel mixture was added to each of five CellCulture Inserts (product number “353096” manufactured by BD Falcon), andthe Cell Culture Inserts were set in wells of a 24-well plate for tissueculture (product number “3820-024” manufactured by AGC TECHNO GLASS CO.,LTD.) with 1 mL of a DMEM medium containing 10% calf serum. In thiscase, the cell concentration in the cell-gel mixture was 1.45×10⁵cells/100 μL. Next, cell culture was carried out in an incubator at 37°C. in the presence of 5% CO₂. Cell growth rates were measured using atrade name “Cell Counting Kit 8” (manufactured by DOJINDO LABORATORIES)on Day 0 (Hour 2), Day 1, Day 3, and Day 5 after the start of culture.As a result, as shown in FIG. 9, the cell growth rates increased inaccordance with a lapse of culture days.

A specific procedure for the measurement of the cell growth ratesdescribed above is as described below. In other words, 1 mL of themedium in the well was exchanged by 1 mL of a fresh medium, 100 μL of aCell Counting Kit 8 solution were added, and the Cell Culture Insert wasset in the well, followed by incubation at 37° C. for 2 hours. After theincubation, 100 μL of the medium permeating the gel in the Cell CultureInsert were transferred to a well of a 96-well plate, and the absorbanceof the medium at 450 nm was measured using a plate reader. Cell growthrates in the respective culture days were determined with the absorbanceof a sample on Day 0 after the start of culture defined as 100.

As seen from the results of Examples 5 to 7, the peptide gel of thepresent invention has biocompatibility and hence may be suitably used asa substrate for cell culture.

Example 8

The modified peptide 1 was dissolved in a sodium carbonate solution toprepare a 0.5 w/v % peptide aqueous solution (final concentration ofsodium carbonate: 4.5 mM). The peptide aqueous solution had a pH in aneutral region. The peptide aqueous solution was subjected tosterilization treatment at 121° C. for 20 minutes using an autoclaveapparatus (product number “MLS 3020” manufactured by SANYO Electric Co.,Ltd.). The mass of a peptide molecule included in the peptide aqueoussolution before or after the sterilization treatment was examined by amatrix-assisted laser desorption/ionization time-of-flight massspectrometry method (MALDI-TOF-MS) using a time-of-flight massspectrometry apparatus (product number “autoflex III” manufactured byBruker). FIG. 10 show the results.

Comparative Example 3

Sterilization treatment and mass spectrometry (MALDI-TOF-MS) werecarried out in the same manner as in Example 8 except that a trade name“PuraMatrix™” (manufactured by 3-D Matrix, Ltd.) was used in place ofthe peptide aqueous solution of the modified peptide 1. FIG. 11 show theresults.

As shown in FIG. 10, the peptide of the present invention issubstantially free of degradation by the sterilization treatment. Thus,the peptide of the present invention may be turned into a sterile statethrough the sterilization treatment. Meanwhile, as shown in FIG. 11, itis understood that the trade name “PuraMatrix™” (manufactured by 3-DMatrix, Ltd.) undergoes peptide degradation through the sterilizationtreatment. This is presumably because the trade name “PuraMatrix™”(manufactured by 3-D Matrix, Ltd.) is an acidic peptide aqueoussolution.

Example 9

The modified peptide 1 was dissolved in a sodium carbonate solution toprepare a 0.8 w/v % peptide aqueous solution (final concentration ofsodium carbonate: 4.5 mM). The resultant peptide aqueous solution wasleft to stand still at 22° C. for 2 hours to form a peptide gel. The gelwas transferred to a laboratory dish (diameter: 6 cm) made of glasswithout any particular care. FIG. 12( a) shows a photograph in thiscase. As shown in FIG. 12( a), the gel included air bubbles and was toohard to uniformly coat the laboratory dish.

The laboratory dish with the gel was placed in a freezer at −20° C. tofreeze the gel. FIG. 12( b) shows a photograph of the frozen gel. Afterthat, the laboratory dish was taken out of the freezer, and the gel wasmelted under a room temperature condition while the laboratory dish wasshaken. The resultant sol was applied to the entire bottom of thelaboratory dish. The laboratory dish was left to stand still in thisstate to reconstruct the gel. Thus, the laboratory dish whose entirebottom was uniformly coated with the peptide gel was obtained. FIG. 12(c) shows a photograph of the laboratory dish.

Example 10

A slide glass whose entire surface was uniformly coated with a peptidegel was obtained in the same manner as in Example 9 except that a slideglass was used in place of the laboratory dish made of glass. FIGS. 13(a), 13 (b), and 13 (c) show a photograph of the gel transferred to theslide glass, a photograph of the frozen gel, and a photograph of theslide glass whose entire surface was uniformly coated with the peptidegel, respectively.

INDUSTRIAL APPLICABILITY

The self-assembling peptide or the like of the present invention may beapplied for regenerative medicine, a drug delivery system, a cosmetic,an artificial vitreous body, a hemostat, an injection for cosmeticsurgery, bone filling, a joint lubricant, a water retention material formoistening, or the like.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1 is a self-assembling peptide of the present invention.SEQ ID NO: 2 is a self-assembling peptide of the present invention.SEQ ID NO: 3 is a modified peptide of the present invention.SEQ ID NO: 4 is a peptide which is not a self-assembling peptide of thepresent invention.SEQ ID NO: 5 is a peptide which is not a self-assembling peptide of thepresent invention.SEQ ID NO: 6 is a self-assembling peptide of the present invention.SEQ ID NO: 7 is a self-assembling peptide of the present invention.SEQ ID NO: 8 is a self-assembling peptide of the present invention.SEQ ID NO: 9 is a self-assembling peptide of the present invention.SEQ ID NO: 10 is a self-assembling peptide of the present invention.SEQ ID NO: 11 is a self-assembling peptide of the present invention.SEQ ID NO: 12 is a modified peptide of the present invention.

1-16. (canceled)
 17. A self-assembling peptide, comprising an amino acidsequence: a₁b₁c₁b₂a₂b₃db₄a₃b₅c₂b₆a₄, wherein a₁ to a₄ each represent abasic amino acid residue; b₁ to b₆ each represent a hydrophobic aminoacid residue; c₁ and c₂ each represent an acidic amino acid residue; andd represents a hydrophobic amino acid residue; wherein at least twoamino acid residues selected from b₃, b₄, and d represent a leucineresidue.
 18. The self-assembling peptide according to claim 17, whereinall of b₁ to b₆ in the amino acid sequence each represent a leucineresidue.
 19. The self-assembling peptide according to claim 17, whereind in the amino acid sequence represents an alanine residue, a valineresidue, a leucine residue, or an isoleucine residue.
 20. Theself-assembling peptide according to claim 17, wherein the basic aminoacid is arginine or lysine.
 21. The self-assembling peptide according toclaim 17, wherein the acidic amino acid is aspartic acid or glutamicacid.
 22. A modified peptide, comprising the self-assembling peptideaccording to claim 17, wherein at least one of the N-terminal aminogroup and the C-terminal carboxyl group is modified, and the modifiedpeptide has a self-assembling ability.
 23. The modified peptideaccording to claim 22, wherein at least one amino acid is introducedinto at least one of the N-terminal amino group and the C-terminalcarboxyl group of the self-assembling peptide.
 24. The modified peptideaccording to claim 22, wherein the modified peptide is acetylated at theN-terminus, amidated at the C-terminus, or both.
 25. A peptide gel,formed from an aqueous solution comprising at least one member selectedfrom the group consisting of the self-assembling peptide according toclaim 17 and a modified peptide, wherein the modified peptide comprisesthe self-assembling peptide, at least one of the N-terminal amino groupand the C-terminal carboxyl group is modified, and the modified peptidehas a self-assembling ability.
 26. The peptide gel according to claim25, wherein the aqueous solution has a pH of 5 to
 9. 27. The peptide gelaccording to claim 25, wherein a total concentration of theself-assembling peptide and the modified peptide in the aqueous solutionis 0.3 to 0.8 w/v %.
 28. The peptide gel according to claim 27, whereinthe peptide gel has an absolute value L (g/s) of an amount of change inload per unit time of 0.03 g/s or more in an approximate straight lineof values measured from the start of compression to between 8 and 10seconds in a compression test carried out at a compression speed of 0.05mm/s using a jig whose tip has a spherical shape with a diameter of 3.2mm and a curvature radius of 1.6 mm under a temperature condition of 22°C.
 29. The peptide gel according to claim 26, wherein the aqueoussolution is subjected to sterilization treatment under a pressurizedcondition at 100° C. or more in advance.
 30. The peptide gel accordingto claim 25, wherein the peptide gel has a visible light transmittancemeasured at an absorbance of 380 nm to 780 nm in a cell having anoptical path length of 10 mm of 90% or more.